Type I (Rituximab, Ofatumumab) antibodies bind CD20 and redistribute it into lipid rafts on the cell membrane, enabling strong complement activation (CDC) but only moderate ADCC. They also induce CD20 internalization (antigen shaving). Type II (Obinutuzumab, Tositumumab) antibodies do not trigger lipid raft clustering, resulting in weak CDC but potent direct cell death (lysosome-mediated) and enhanced ADCC. Obinutuzumab is additionally glycoengineered (afucosylated Fc) for maximal FcγRIIIa-mediated NK cell engagement, giving it the strongest ADCC activity of all approved anti-CD20 antibodies.

CD20 is a four-transmembrane protein with very small extracellular loops (~44 amino acids for the larger loop). The therapeutic antibody epitopes span these loops, which adopt their native conformation only when embedded in the lipid bilayer. Soluble CD20 ECD fragments are difficult to produce and may not fully recapitulate the native epitope. For binding assays where conformational accuracy is critical (e.g., epitope mapping, biosimilar comparability), cell-based binding assays using CD20⁺ cell lines (Raji, Daudi, WIL2-S) are often preferred over recombinant protein ELISA.

For CDC: use Daudi or WIL2-S cells as CD20⁺ targets. Add serial dilutions of Rituximab biosimilar vs reference, then add normal human serum as complement source (10-20%). Measure cell lysis after 2-4 hours by CellTiter-Glo or PI uptake. For ADCC: use WIL2-S or Raji cells as targets with either primary NK cells (CD56⁺ isolation from PBMCs) or an ADCC reporter cell line (Jurkat-FcγRIIIa-NFAT-Luc). Add Rituximab biosimilar vs reference and measure killing (LDH release) or reporter luminescence after 4-6 hours. Both assays require dose-response curve parallelism and relative potency within 80-120%.

Antigen shaving (trogocytosis) is a process where FcγR-expressing cells (monocytes, macrophages, NK cells) strip CD20-antibody complexes from the target B-cell surface without killing the cell. This reduces CD20 surface density, potentially leading to treatment resistance. Type I antibodies (Rituximab) are more susceptible to shaving than Type II antibodies (Obinutuzumab). Antigen shaving is measured by flow cytometry: incubate CD20⁺ cells with anti-CD20 antibody + monocytes, then quantify residual CD20 on surviving B cells. This is relevant for biosimilar comparability — shaving kinetics should be similar between biosimilar and reference.

Anti-CD20 B-cell depletion has expanded far beyond lymphoma. Rituximab is approved or used off-label for rheumatoid arthritis, ANCA vasculitis, pemphigus vulgaris, and immune thrombocytopenia. Ocrelizumab (Ocrevus) and Ofatumumab (Kesimpta) are approved for multiple sclerosis. The rationale is that pathogenic B cells — as antigen-presenting cells, cytokine producers, and autoantibody sources — drive autoimmune pathology. B-cell depletion removes these functions while antibody-producing plasma cells (CD20⁻) are spared, maintaining existing humoral immunity.